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Lesson Outline Overview of Recombinant DNA and Gene Cloning Objectives for Gene Cloning Part 1 The Gene Cloning Process Why clone genes? DNA is cloned from DNA Genes are Connected to Other Genes Gene Cloning Requires Knowledge of the Gene’s Unique Characteristics. Plasmids do the “Tricking” Cloning with LacZ and X-gal plasmids Step One: Make Sticky Ends Step Two: Make Recombinant Plasmids Step Three: Transformation of E. coli Step Four: Selecting Bacteria with Recombinant Plasmids Step Five: Making Libraries of Genes Genomic Libraries cDNA library Gene Libraries with Big Pieces Lesson Media Objects Making a Recombinant Plasmid Bacteria Transformation Screening a DNA Library Making a Gene Library

Gene Cloning Part 1: The Mechanics of Recombinant DNA Rate Me 1 2 3 4 5 Step Two: Make Recombinant Plasmids If DNA ligase was added to the tube of cut DNA, the sugar-phosphate backbone bonds would be sealed and the two cut linear DNA molecules would become one circular recombinant plasmid. This plasmid will have an unaltered Amp antibiotic resistance gene and an inactivated LacZ gene with the insertion. The inactivation of one gene on the plasmid while leaving the other gene unchanged is a key requirement for gene cloning. Fig.9: Foreign DNA with gene of interest (A, black)and plasmid (B) cut with same restriction enzyme Fig.10: Cut foreign DNA and plasmid mixed (A), ligase enzyme added (B) and recombinant plasmids made (C). The formation of a recombinant plasmid is taking place in a test tube. The cutting and ligating enzymes will work if the right combination of DNA and enzymes is added even if the gene cloner cannot see what is happening inside the tube. Because the resealing relies on chemistry and the chance encounter of sticky ends, a number of different kinds of recombinant plasmids can be made. One common result is the sticky ends from the same plasmid molecule coming together. This will give the same plasmid sequence as the original and is called the nonrecombinant plasmid. Thus the test tube of the gene cloner will contain a combination of recombinant and nonrecombinant plasmids. Their next step is to introduce these plasmids into E.coli bacteria cells. View ’Making a Recombinant Plasmid’ Animation Comments Be the first to write a comment... Add Comment

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This project was supported in part by the National Research Initiative Competitive Grants CAP project 2011-68002-30029 from the USDA National Institute of Food and Agriculture, adminstered by the University of California-Davis and by the National Science Foundation (NSF), Division of Undergraduate Education, National SMETE Digital Library Program, Award #0938034, administered by the University of Nebraska. Any opinions, findings, conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the views of the USDA or NSF.
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